Versatile single-step-assembly CRISPR/Cas9 vectors for dual gRNA expression

CRISPR/Cas9 technology enables efficient, rapid and cost-effective targeted genomic modification in a wide variety of cellular contexts including cultured cells. Some applications such as generation of double knock-outs, large deletions and paired-nickase cleavage require simultaneous expression of...

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Published in:PloS one 2017-12, Vol.12 (12), p.e0187236-e0187236
Main Authors: Adikusuma, Fatwa, Pfitzner, Chandran, Thomas, Paul Quinton
Format: Article
Language:English
Subjects:
Animals
Biology and life sciences
Biotechnology
Cells, Cultured
Cleavage
Cloning
Cloning, Molecular
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR
Deoxyribonucleic acid
DNA
Embryo cells
Embryonic stem cells
Embryonic Stem Cells - metabolism
Engineering and Technology
Expression vectors
Gene expression
Genetic recombination
Genetic Vectors
Genomes
gRNA
Inserts
Laboratories
Mice
Multiplexing
Muscular dystrophy
Nuclease
Oligonucleotides
Physiological aspects
Plasmids
Polymerase Chain Reaction
Research and analysis methods
RNA processing
RNA, Guide, CRISPR-Cas Systems - genetics
Stem cell transplantation
Stem cells
Transfection
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cited_by cdi_FETCH-LOGICAL-c692t-79427f3995bbf8c338fcc832b638d0891f0d2f264c6b0fa82895868d5de76d333
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description CRISPR/Cas9 technology enables efficient, rapid and cost-effective targeted genomic modification in a wide variety of cellular contexts including cultured cells. Some applications such as generation of double knock-outs, large deletions and paired-nickase cleavage require simultaneous expression of two gRNAs. Although single plasmids that enable multiplex expression of gRNAs have been developed, these require multiple rounds of cloning and/or PCR for generation of the desired construct. Here, we describe a series of vectors that enable generation of customized dual-gRNA expression constructs via an easy one-step golden gate cloning reaction using two annealed oligonucleotide inserts with different overhangs. Through nucleofection of mouse embryonic stem cells, we demonstrate highly efficient cleavage of the target loci using the dual-guide plasmids, which are available as Cas9-nuclease or Cas9-nickase expression constructs, with or without selection markers. These vectors are a valuable addition to the CRISPR/Cas9 toolbox and will be made available to all interested researchers via the Addgene plasmid repository.
doi_str_mv 10.1371/journal.pone.0187236
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Some applications such as generation of double knock-outs, large deletions and paired-nickase cleavage require simultaneous expression of two gRNAs. Although single plasmids that enable multiplex expression of gRNAs have been developed, these require multiple rounds of cloning and/or PCR for generation of the desired construct. Here, we describe a series of vectors that enable generation of customized dual-gRNA expression constructs via an easy one-step golden gate cloning reaction using two annealed oligonucleotide inserts with different overhangs. Through nucleofection of mouse embryonic stem cells, we demonstrate highly efficient cleavage of the target loci using the dual-guide plasmids, which are available as Cas9-nuclease or Cas9-nickase expression constructs, with or without selection markers. 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subjects Animals
Biology and life sciences
Biotechnology
Cells, Cultured
Cleavage
Cloning
Cloning, Molecular
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR
Deoxyribonucleic acid
DNA
Embryo cells
Embryonic stem cells
Embryonic Stem Cells - metabolism
Engineering and Technology
Expression vectors
Gene expression
Genetic recombination
Genetic Vectors
Genomes
gRNA
Inserts
Laboratories
Mice
Multiplexing
Muscular dystrophy
Nuclease
Oligonucleotides
Physiological aspects
Plasmids
Polymerase Chain Reaction
Research and analysis methods
RNA processing
RNA, Guide, CRISPR-Cas Systems - genetics
Stem cell transplantation
Stem cells
Transfection
title Versatile single-step-assembly CRISPR/Cas9 vectors for dual gRNA expression
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